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From 2010.igem.org

Contents 1 Andreas 1.1 IgG protease activity assay 1.1.1 Results 1.1.2 Assay restart

Andreas

IgG protease activity assay

Continued the protocol and read the wavelength at 450 nm.

Blank: 80 μl PBS + 100 μl SureBlue™ peroxidase substrate + 100 μl 1 M HCl.

Sample loading
Extract dil.	Ab dil.	1:500						1:1000
		1	2	3	4	5	6	7	8	9	10	11	12
1.5x105	A	Ind. IgGp		Ind. SOD		Unind. IgGp		Ind. IgGp		Ind. SOD		Unind. IgGp
1.5x108	B	Ind. IgGp		Ind. SOD		Unind. IgGp		Ind. IgGp		Ind. SOD		Unind. IgGp
–	C	PBS 1:500 (cont)		PBS 1:1000 (cont)		Blank	–	–	–	–	–	–	–

Results

A450 measurements
	1	2	3	4	5	6	7	8	9	10	11	12
A	0.007	0.006	0.008	0.008	0.006	0.007	0.006	0.007	0.011	0.011	0.005	0.006
B	0.096	0.099	0.054	0.056	0.060	0.062	0.035	0.033	0.047	0.049	0.048	0.050
C	0.004	0.006	0.004	0.003	0.000	–	–	–	–	–	–	–

Results from the different antibody dilutions are somewhat contradictory. The 1.5x10⁸/1:500 samples indicate a higher IgG protolysis activity for the induced IgGp sample, followed by the uninduced IgGp and induced SOD, which would be the expected results from a functional IgG protease (IdeS). However, for the corresponding 1:1000 antibody dilution the results are almost opposite.
Furthermore, the 1.5x10⁵ dilutions seem too low to give any recordable and reliable results.

The experiment will be repeated with the 1.5x10⁸ extracts and 1:500 as well as 1:250 antibody dilutions, so see if the results are reproducible.

Assay restart

Assay restarted as described above, with duplicate samples.